ES2663272T3 - Procedure for the preparation of L-phenylephrine using an alcohol dehydrogenase - Google Patents

Abstract

Process for the preparation of substituted optically active alcohols of the formula IV ** Formula ** in which Cyc represents a mono or polynuclear, saturated or unsaturated, carbocyclic or heterocyclic ring, optionally substituted one or more times, which carries the less one free hydroxyl group, and R1 and R2 independently of each other represent H or an alkyl radical optionally substituted one or more times; or salts of this compound; in each case in the form of a pure stereoisomer or as a mixture of stereoisomers, in which a) a ketone of the formula I ** Formula ** in which Cyc has the meanings indicated above, reacts with a halogenating agent in the presence of a aliphatic alcohol to give a halogenated compound of formula II ** Formula ** in which Cyc has the meanings indicated above and Hal represents a halogen atom; b) the compound of formula II thus obtained is enzymatically reduced using an enzyme chosen from alcohol dehydrogenases (ADH) (EC 1.1.1.1), until giving the alcohol of formula III ** Formula ** in which Cyc and Hal have the meanings indicated above; and c) the alcohol of the formula III thus obtained reacts with an amine of the formula HNR1R2, in which R1 and R2 have the meanings indicated above; to give the compound of formula IV.

Description

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DESCRIPTION

Procedure for the preparation of L-phenylephrine using an alcohol dehydrogenase

The present invention relates to a multi-stage process for the preparation of optically active substituted alcohols, comprising a step of synthesis with enzymatic catalysis, catalyzed by an alcohol dehydrogenase. In particular, the process according to the invention is suitable for the preparation of phenylephrine, ie 3 - [(1R) -1-hydroxy-2-methylamino-ethyl] -phenol.

Basis of the invention:

Phenylephrine is an active pharmacological principle of the sympathomimetic group and has agonistic activity in the a1-adrenergic receptor. It is structurally equal to adrenaline, except for the missing 3-hydroxyl group and finds application primarily as a local vasoconstrictor. As an active ingredient in nasal drops, it acts as a decongestant in the mucosa. In eye drops it also acts as mydriatic, therefore it leads to dilation of the pupils.

The preparation of phenylephrine is already described in the literature. Apart from the numerous processes, which produce the desired product as a racemate and then by excision with a suitable chiral auxiliary agent transform the product, methods for stereoselective synthesis are considered preferred, since by them non-economic destruction can be avoided of the wrong enantiomer that can reach up to 50%.

Preparation processes known from the state of the art of L-phenylephrine hydrochloride include asymmetric hydrogenation of the N-benzyl-N-methyl-2-amino-m-benzyloxyacetophenone hydrochloride according to Tetrahedron Letters 30 (1989 ), 367- 370, or Chem. Pharm. Bull. 43 (5) (1995) 738-747.

Achiwa et al. described in Tetrahedron Letters 30 (1989), 367-370 asymmetric hydrogenation of 3- benzyloxy-2- (N-benzyl-N-methyl) -aminoacetophenone hydrochloride as substrate, with hydrogen in the presence of [Rh (COD) Cl] 2 / (2R, 4R) -4- (dicyclohexylphosphino) -2- (diphenylphosphino-methyl) -N-methyl-aminopyrrolidine as catalyst. Immediately after filtration and concentration of the reaction mixture, the benzyl nitrogen protecting group is cleaved and obtained as a phenylephrine product. In this regard, apart from the L enantiomer, the D enantiomer has a proportion of at least 7.5% as a contaminant (85% ee). For the reaction, the catalyst, referred to the substrate, must be used in a molar ratio of 1: 2000. The disadvantage of this process is essentially that the L-phenylephrine obtained cannot be economically purified at a purity of at least 98% ee necessary for the application as a medicine.

In Chem. Pharm. Bull. 43 (5) (1995) 738-747 a molar ratio of substrate to catalyst of about 1000 is indicated as preferred for asymmetric hydrogenation. However, despite the use of large amounts of catalyst in the asymmetric reaction stage , the product cannot be prepared with sufficient purity as an L enantiomer for pharmaceutical purposes, without expensive purification procedures, but is accessible only as a mixture with a relatively high proportion of enantiomer D as an impurity. Also the relatively long reaction time of the asymmetric hydrogenation stage, of approximately 20 hours, represents precisely for the manufacture of L-phenylephrine on an industrial scale, a reaction stage very elaborate in equipment and expensive, with a non-negligible safety risk .

The procedure taught in WO 00/43345 satisfies some of the conditions mentioned for a reasonably economical manufacture of L-phenylephrine hydrochloride, although the use of protective groups cannot be waived here, so the process becomes inexpensive. In addition, also after this procedure for the stereoselective stage, the desired product arises only with 93% ee, so that here also an expensive purification must follow.

In Tetrahedron Asymmetry 18 (15) (2007) 1799-1803 the activity and enantioselectivity of the YMR226c dehydrogenase of S. cerevisiae was investigated with a series of ketones. It is described that YMR226c dehydrogenase catalyzes enantioselective reductions of aryl substituted acetophenones, a-chloracetophenones, aliphatic ketones and a- and p-ketoesters.

WO 2005/108590 describes a process for the preparation of optically active alcohols, by enantioselective reduction, using Azoarcus sp EbN1 phenylethanol dehydrogenase.

Brief description of the invention:

Therefore, it is an objective of the present invention to provide a novel process for the preparation of optically active alcohols, such as L-phenylephrine, which is more economically executable, compared to the prior art. In particular, such an improved procedure should be handled without use.

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of protective groups and should have a high spatial selectivity.

Surprisingly, the above objective could be achieved by providing a process for the preparation of optically active substituted alcohols of formula IV, as follows:

image 1

in which

Cyc represents a mono or polynuclear ring, saturated or unsaturated, carbocyclic or heterocyclic, where appropriate substituted once or several times, which carries at least one free hydroxyl group, and

R1 and R2 independently of each other represent H or an alkyl radical, where appropriate substituted once or several times, or salts of this compound,

in each case in the form of pure stereoisomer or as a mixture of stereoisomers, in which

a) a ketone of the formula

image2

in which Cyc has the meanings indicated above,

in the presence of an aliphatic alcohol, it reacts with a halogenating agent to give a halogenated compound of the formula II

image3

in which Cyc has the meanings indicated above and Hal represents a halogen atom,

b) the compound thus obtained from formula II is reduced enzymatically using an enzyme chosen from alcohol dehydrogenases (ADH) (E.C. 1.1.1.1), to give alcohol of formula III

OH

image4

Hal

in which Cyc and Hal have the meanings indicated above, and

c) the alcohol of the formula III thus obtained reacts with an amine of the formula HNR1 R2,

in which R1 and R2 have the meanings indicated above,

until giving the compound of the formula IV.

In summary, the present invention makes possible in particular a surprisingly advantageous process for the preparation of the active ingredient phenylephrine (3 - [(1R) -1-hydroxy-2-methylamino-ethyl] -phenol; 4). This preferred embodiment can be represented by the following reaction scheme:

Scheme 1

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12 3 4

In this regard, one of the two key steps is the selective chlorination of the 3′-hydroxyacetophenone side chain (3- hAP, 1) to 3’-hydroxy-2-chloroacetophenone (HCAP, 2).

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The second key step concerns the enantioselective reduction of HCAP (2) to (R) -3- (2-chloro-1-hydroxyethyl) -phenol (HCPE, 3), using an enzyme, and specifically an alcohol dehydrogenase (ADH) .

The procedure supplied according to the invention clearly differs in some essential points from the state of the art discussed above.

That is, the totality of the synthesis occurs well without the use of protective groups, whereby the procedure is more economical compared to the prior art. This is especially surprising and unexpected for the first stage.

By using alcohol dehydrogenase as a hydrogenation catalyst, cheaper access to (R) -3- (2-chloro-1-hydroxyethyl) -phenol (HCPE, 3) with high optical purity is possible. It is not formed in amounts worth mentioning the unwanted enantiomer. (The% ee values for the desired enantiomer are in the range of> 98%, such as> 99% to about 100%, such as about 99.9%).

The reaction can be executed (without being limited by it) in addition to a two-phase system of organic solvent and water, which also makes a more economical job possible. In this regard, a more complete transformation of acetone to the desired alcohol is possible. The processing of the mixture is particularly beneficial because of the biphasic nature, because the product is separated from the catalyst (protein) residues by extraction. In addition, the use of the organic phase reduces the exposure of the biocatalyst with the low molecular weight phenolic ketone, whereby inactivation and / or inhibition of the catalyst is avoided.

Description of the figure:

Figure 1 shows the nucleic acid sequence or amino acid sequence of the phenylethanol dehydrogenase of (Azoarcus sp) Aromatoleum aromaticum EbN1 (SEQ ID NO: 1 or 2).

Detailed description of the invention:

1. Preferred embodiments

A first objective of the invention relates to a process for the preparation of optically active substituted alcohols of the formula IV

image6

in which

Cyc represents a mono or polynuclear ring, in particular mononuclear, with 4 to 7 members, in particular 5 or 6 members, saturated or unsaturated, in particular unsaturated, especially aromatic, carbocyclic or heterocyclic, in particular carbocyclic, which carries at least a free hydroxyl group, and if necessary is substituted once or several times, in which in the case of a 6-membered ring the hydroxyl group (s) are in particular in a meta position relative to the side chain of Cyc which carries amino groups; Y

R1 and R2 independently of each other represent H or identical or different alkyl radicals, if necessary substituted once or several times;

or of salts of this compound, for example acid addition salts of in particular inorganic acids, such as HCl; in each case in the form of pure stereoisomer, such as form (R) or (S), or as a mixture of stereoisomers, such as racemates,

in which

a) a ketone of the formula I

image7

in which Cyc has the meanings indicated above,

reacts in the presence of a halogenated aliphatic alcohol, such as in particular chlorinated, and particularly in particular with sulfuryl chloride, to give a halogenated compound, in particular chlorinated, of the formula II

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in which Cyc has the meanings indicated above and Hal represents a halogen atom, such as F, Br or in particular Cl;

b) the compound of the formula II thus obtained, if necessary after prior isolation or enrichment, is reduced enzymatically using an enzyme chosen from among alcohol dehydrogenases (ADH) (EC 1.1.1.1) to give the alcohol of the formula III

OH

image9

Hal

in which Cyc and Hal have the meanings indicated above; Y

c) the alcohol of the formula III thus obtained, if necessary after isolation or prior enrichment, reacts with an amine of the formula HNR1R2, in which R1 and R2 possess the meanings indicated above, until the compound of the formula IV is given and this is isolated in the case of the reaction mixture, in the case of pure stereoisomer.

The ketones of the formula I above used for synthesis are known per se compounds and accessible by application of known organic synthesis procedures in general.

In particular, the reaction of step a) occurs in the presence of 1 to 10, 2 to 8 or 3 to 5 molar equivalents of aliphatic alcohol per mole of ketone of formula I.

Suitable aliphatic alcohols are in particular mono- or polyols with 1 to 6, in particular 1 to 4 carbon atoms and 1 to 5, in particular 1 to 3 hydroxyl groups, in particular monools with 1 to 4 carbon atoms, as per example methanol, ethanol, n-propanol, n-butanol; or long chain monooles, such as n-pentanol and n-hexanol, or polyols, such as propanediol, butane-1,4-diol, pentane-1,5-diol, hexane-1,6-diol or pentane-1,3 , 5-triol; and isomeric forms of these mentioned alcohols.

The chemical reaction in step c) can occur in particular in solution in an open or cyclic chain ether. Suitable ethers are in particular MTBE, methyl-THF, dioxane and especially THF.

Step b) of the reaction according to the invention is catalyzed by at least one enzyme, chosen from alcohol dehydrogenases (ADH) (E.C. 1.1.1.1). In this regard, they are the ADHs, chosen for example from among alcohol dehydrogenases of microorganisms of the genus Aromatoleum (Azoarcus), in particular of the bacterium Aromatoleum Aromaticum EbN1.

For example, the enzyme for the execution of step b) is chosen from among enzymes that possess a polypeptide sequence that is chosen from

(i) SEQ ID NO: 2 or

(ii) sequences, in which up to 25%, such as 1 to 24%, 2 to 20%, 3 to 15% or 4 to 10%, of the amino acid radicals are modified with respect to SEQ ID NO: 2 by addition , elimination, insertion, substitution, investment or a combination of them, and / or that still exhibit activity of at least 50%, such as at least 60, 70, 80, 90, 95, 96, 97, 98, 99, 100 or more than 100%, such as 1 to 20 times, or 2 to 10 times or 3 to 5 times, of the enzyme activity of an enzyme according to SEQ ID NO: 2.

According to another modification, the reaction of step b) occurs by adding reduction equivalents, in particular NADH or NADPH, and if necessary under simultaneous or temporary displaced regeneration of the reduction equivalent consumed for the reaction.

To this end, regeneration can occur in a manner known per se by enzymatic, electrochemical or electroenzymatic routes (Biotechnology Progress, 2005, 21, 1192; Biocatalysis and Biotransformation, 2004, 22, 89; Angew. Chem Int. English edition, 2001 , 40, 169; Biotechnol Bioeng, 2006, 96, 18; Biotechnol Adv., 2007, 25, 369; Angew.Chem Int. English edition, 2008, 47, 2275; Current Opinion in Biotechnology, 2003, 14, 421; Current Opinion in Biotechnology, 2003, 14, 583). In particular, regeneration occurs enzymatically, and the regenerating enzyme is chosen from ADH (EC.1.1.1.1) and dehydrogenases other than ADH, such as glucose dehydrogenases (EC 1.1.1.47), formate dehydrogenases (EC 1.2.1.2 or EC) 1.2.1.43), and

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phosphitodehydrogenases (EC 1.20.1.1) and preferably in the presence of a so-called "slaughter alcohol", such as butane- or pentane-2-ol, which is consumed in the enzymatic regeneration of the reduction equivalent, that is, it is oxidized.

In particular, the reaction of step b) can occur either in the presence of a microorganism which expresses ADH naturally or recombinantly, or in the presence of a fraction derived therefrom that contains ADH, that is, obtained from the cells, or extract. cell obtained from the cells, or in the presence of the pure or essentially pure enzyme. The enzyme used according to the invention (in pure form, in enriched form, or as a cell extract containing the enzyme) is also used in a manner known per se, dissolved, dispersed or immobilized on a support.

For example, the reaction of stage b) occurs in the presence of a microorganism that is chosen from among the bacteria of the Enterobacteriaceae, Pseudomonadaceae, Bacillaceae, Rhizobiaceae, Lactobacillaceae, Streptomycetaceae, Rhodococcaceae, Rhodociclaceae and Nocardiaceae families, or in the presence of a fraction or an extract derived from them. Examples of suitable genera include in particular Escherichia, Streptomyces, Corynebacterium and Bacillus. It is an example of a suitable type in particular E. coli.

In particular, the microorganism can be a recombinant microorganism, which is transformed with a nucleic acid fragment, which codes for an ADH according to the above definition. If necessary, the recombinant microorganism used can additionally express an exogenous or exogenous dehydrogenase other than ADH according to the above definition, to promote the regeneration of the cofactor.

In another modification, the reaction of step b) can be performed in a two-phase liquid reaction medium. For this purpose, for example, an aqueous-organic reaction medium is used, in which both the reagent of the formula II and also the product of the formula III dissolve better in the organic phase than in the aqueous phase, such as a phase aqueous-ethereal, or for example water / heptane and water / hexane phases.

Another objective described herein refers to a process for the preparation of a compound of the general formula II,

image10

in which Cyc and Hal have the meanings indicated above, in which a ketone of the formula I is halogenated, in particular chlorinated

image11

in which Cyc has the meanings indicated above,

in particular in the presence of an aliphatic alcohol, with a suitable halogenating agent, such as in particular sulfuryl chloride, to the compound of the halogenated formula II, in particular chlorinated.

In this regard, the reaction of step a) occurs in particular in the presence of 1 to 10, such as 2 to 8 or 3 to 5, molar equivalents of alcohol per mole of ketone of formula I.

Another objective described herein refers to a process for the preparation of a compound of the formula III

OH

image12

Hal

in which Cyc and Hal have the meanings indicated above; in which a compound of the general formula II

image13

Hal

in which Cyc and Hal have the meanings indicated above, it is enzymatically reduced to alcohol

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of the formula III. In this regard, the enzymatic reaction is conducted as defined above.

According to the invention, Cyc represents in particular a 4, 5 or 6 membered mononuclear, carbocyclic or heterocyclic aromatic ring, which carries at least one HO group, such as in particular a 3-hydroxyphenyl radical. Hal represents in particular a chlorine atom.

Another object of the invention relates to the use of an alcohol dehydrogenase according to the above definition or a microorganism that produces this enzyme according to the above definition, for the preparation of compounds of the formula IV, in particular for the preparation of (3 - [(1R) -1-hydroxy-2-methylamino-ethyl] -phenol).

2. Definitions

2.1 General concepts

If no other data is given, then the following general meanings are valid:

"Optically active" are compounds according to the invention with at least one center of asymmetry in the molecule.

A "free hydroxyl group" means according to the invention that it is not present as a derivative, such as as an ester or ether group.

Products of pure stereoisomer or pure enantiomer are understood as (3 - [(1R) -1-hydroxy-2-methylaminoethyl] -phenol or (R) -3- (2-chloro-1-hydroxyethyl) -phenol, enantiomers according to the invention showing enantiomer enrichment, in particular in the process according to the invention, enantiomer purities of at least 90% ee, preferably at least 95% ee, particularly preferably preferably at least 98% ee, very particularly preferably at least 99% ee or more.

The "enantiomer purity" is defined by the parameters

ee% = [XA-XB] / [Xa + Xb] * 100,

in which XA and XB represent the molar fraction of the A or B enantiomers.

A reaction occurs "enzymatically" in the presence of pure enzymes, enriched enzymes or whole cells.

2.2 Special chemical meanings

"Mono or polynuclear" radicals are radicals that comprise one or more cyclic groups, in which, in the case of polynuclear radicals, these cyclic groups can be linked to each other directly or by current or mutually condensed bridge groups.

"Carbocyclic" radicals exclusively comprise carbon atoms in the ring; "heterocyclic" radicals further comprise one or more, such as 1, 2 or 3, same or different ring heteroatoms, such as N, O or S.

These carbon or heterocyclic rings comprise in particular 3 to 12, preferably 4, 5 or 6 carbon atoms in the ring. As examples, there may be mentioned cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, the analogs thereof unsaturated once or several times, such as cyclobutenyl, cyclopentenyl, cyclohexenyl, cycloheptenyl, cyclohexadienyl, cycloheptadienyl; as well as heterocyclic radicals with 5 to 7 members, saturated or with one or more unsaturations, with 1 to 4 heteroatoms that are chosen from O, N and S, in which the heterocycle may be condensed with another heterocycle or carbocycle . In particular they are mentioned as heterocyclic radicals, derivatives of pyrrolidine, tetrahydrofuran, piperidine, morpholine, pyrrole, furan, thiophene, pyrazole, imidazole, oxazol, thiazole, pyridine, pyran, pyrimidine, pyridazine, pyrazine, coumarone, indole and quinoline.

Non-limiting examples of suitable "substituents" are those chosen from halogen, OH, -SH, -NO2, small alkyl, small alkenyl, small alkoxy and aryl.

"Halogen" represents fluorine, chlorine, bromine or iodine, in particular fluorine, bromine or chlorine.

"Small alkyl" represents straight or branched chain alkyl radicals with 1 to 6 C atoms, such as methyl, ethyl, i- or n-propyl, n-, i-, sec.- or tert.-butyl, n-pentyl or 2-methylbutyl, n-hexyl, 2-methyl-pentyl, 3-methyl-pentyl, 2-ethyl-butyl.

"Small alkenyl" represents the analogs of the alkyl radicals mentioned above, unsaturated once or several times, preferably unsaturated once or twice with 2 to 6 carbon atoms, in which the double bond

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It can be in any position of the carbon chain.

"Small alkoxy" represents the oxygen-terminated analogs of the above alkyl radicals.

"Aryl" represents a mono or polynuclear aromatic radical, preferably mono or binuclear, if substituted, in particular represents phenyl or represents a naphthyl attached at any ring position, such as 1- or 2- naphthyl. These aryl radicals may, if appropriate, carry 1 or 2 identical or different substituents, chosen from halogen, small alkyl, small alkoxy according to the above definition, or trifluoromethyl.

Examples of suitable Cyc radicals are phenyl, naphthyl, 2-thienyl, 3-thienyl; 2-furanyl, 3-furanyl; 2-pyridyl, 3-pyridyl or 4-pyridyl; 2-thiazolyl, 4-thiazolyl or 5-thiazolyl; 4-methyl-2-thienyl, 3-ethyl-2-thienyl, 2-methyl-3-thienyl, 4-propyl-3-thienyl, 5-n-butyl-2-thienyl, 4-methyl-3-thienyl, 3-methyl-2-thienyl; 3-Chloro-2-thienyl, 4-bromo-3-thienyl, 2-iodo-3-thienyl, 5-iodo-3-thienyl, 4-fluorine-2-thienyl, 2-bromo-3-thienyl, and 4 -chloro-2-thienyl, which additionally carry at least one hydroxyl substituent on the ring.

3. Special modifications to the procedure according to the invention

Other conditioning of the invention will be explained below, by reference to the multi-stage reaction represented in the above scheme 1. Starting from it are the modifications of this procedure specifically described in the field of human capacity.

3.1 Selective chlorination of 3’-hydroxyacetophenone side chain

The main use of sulfuryl chloride for chlorination in ketones is known per se and is described for example in D.P. Wyman et al., J. Org. Chem. Vol. 29, 1964, pages 1956 to 1960.

US 4,310,702 and D. Masilamani et al., J. Org. Chem., Vol. 46, 1981, pages 4486 to 4489 report that the use of sulfuryl chloride for chlorination of ketones generally leads to a mixture of chlorinated ketones once and several times and thereby to unwanted by-products. To solve the problem, the documents teach the use of alcohols or ethers as moderators. In addition, this publication teaches the reaction of phenol with sulfuryl chloride which leads first to the corresponding sulfonic acid ester and then to different chlorophenols. Also US 5,710,341, which refers to the preparation of a-chloroalkylaryl ketones by chlorination of the corresponding ketone with sulfuryl chloride, teaches the use of aliphatic alcohols to increase the selectivity to the desired product, therefore to the chlorinated ketone once in a.

It was now surprisingly found that under the conditions taught in US 5,710,341 of the reaction of 3-hydroxyacetophenone which is to be used advantageously for the synthesis of phenylephrine, a chlorination leads almost exclusively to the corresponding a-chloroalkylaryl ketones. In this regard, 1-10 equivalents of an alcohol (C1-C10) are added to the reaction for the selectivity modulation, with between 3 and 5 equivalents of the alcohol being particularly preferred. In addition, the reaction is carried out in an inert solvent under the reaction conditions such as aromatic compounds, ethers, esters and halogenated solvents, which are not miscible in water. The embodiment in esters and halogenated solvents is preferred, particularly preferably in ethyl acetate or dichloromethane.

image14

1 2

This circumstance is surprising to the expert, since a transformation of the phenolic functionality present in the molecule, analogously to the way taught by D. Masilamani allows the formation of the corresponding expected chlorophenols. In this regard, the transformation can be conveniently executed without the use of a protective group.

3.2. Enantioselective hydrogenation of 3’-hydroxy-2-chloroacetophenone

The reduction of 2 is catalyzed by an enzyme. It is the alcohol dehydrogenase EbN1 of (Azoarcus sp.) Aromatoleum aromaticum EbN1, which in the special case is prepared recombinantly in Escherichia coli.

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It is known that dehydrogenases are suitable as biocatalysts for the preparation of optically active hydroxy compounds. They are well characterized biocatalysts, which are already used in a series of technical processes (Angew. Chem. Int. Ed., 2004, 43, 788; Tetrahedron, 2004, 60, 633; Chiral catalysis - asymmetric hydrogenation supplement to Chemistry Today, 2004 , 22, 26; Current Opinion in Chemical Biology, 2004, 8, 120; Organic Process Research & Development, 2002, 6, 558; Tetrahedron: Asymmetry, 2003, 14, 2659; Chiral catalysis - asymmetric hydrogenation supplement to Chemistry Today, 2004 , 22, 43).

Dehydrogenases transform ketones or aldehydes into the corresponding secondary or primary alcohols; In principle, the reaction is reversible. They catalyze the enantioselective transfer of hydride to the prochemical C atom of the carbonyl compound.

The hydride ions are called cofactors, such as NADPH or NADH (reduced nicotinamide adenine nucleotide phosphate or reduced nicotinamide adenine nucleotide). Since they are very expensive compounds, they are added to the reaction only in catalytic amounts. The reduced cofactors are regenerated during the reaction by a second redox reaction that takes place simultaneously. Depending on the thermodynamic and kinetic conditions of the entire reaction, there may be cheap secondary alcohols (called "sacrificial alcohols") such as i-propanol as the final donor of reaction hydrides, as is known from the reaction from Meerwein-Ponndorf-Verley. Frequently, ketone reduction and sacrificial alcohol oxidation of the same biocatalyst (substrate coupling) can be performed.

Alternatively, a second catalyst can be used to regenerate the consumed cofactors. Known examples of this are formate dehydrogenase, glucose dehydrogenase or phosphite dehydrogenase, which from the oxidation of formate, glucose or phosphite transfer hydride ions from NAD or NADP. (Biocatalysis and Biotransformation, 2004, 22, 89; Applied Microbiology and Biotechnology, 1997, 48, 699; Bioscience Biotechnology and Biochemistry, 1998, 62, 167; Methods Enzymol., 1987, 136, 9; Ann. NYAcad. Sci. , 1984, 434, 91; FEBS Journal, 2005, 272, 3816; Applied Microbiology and Biotechnology, 2003, 61, 133).

The reaction reduction equivalents considered here come either from i-propanol (or another secondary called "sacrificial alcohol") that is oxidized to ketone, or glucose, which in a parallel reaction is oxidized to gluconolactone. While it is possible to oxidize many sacrificial alcohols by the same enzyme, which also executes the reduction of 2 to R-3, for glucose oxidation glucose dehydrogenase must be fed as the second enzyme.

Alternatively, instead of glucose dehydrogenase, another regeneration system can also be used, such as phosphite dehydrogenase (Biotechnol Bioeng, 2006, 96, 18) or an electrochemical cofactor regeneration (Angew. Chem Int.Ed Engl., 2001,40, 169), (Angewandte Chemie Int.Ed.Engl., 1999, 29, 388).

Suitable biocatalysts for the preparation of R-3 are already described in the following BASF SE company patent documents: (DE 2004022686, EP 2005004872, WO 2005108590) or (EP 06123814, WO2008055988 A3).

3.3 Preparation of L-phenylephrine

The termination of this novel process for the preparation of L-phenylephrine and its salts forms the reaction of component 3, obtained after reduction, with methylamine to give the desired product.

image16

3. 4

This is successful in many different inert solvents under the reaction conditions, such as water, alcohols or ethers. In this regard, ethers in which the starting material 3 dissolves in a high proportion are preferred in order to work in reasonably economical concentrations. For this, THF is particularly preferred here. After the reaction, L-phenylephrine can be obtained as a base and in the form of its salts, for example but not exclusively in accordance with the procedure taught in WO 00/43345.

4. Other conditioning of the invention

4.1 Alcohol dehydrogenases

The enzyme used is chosen from alcohol dehydrogenases (E.C. 1.1.1.1). According to the invention, it is

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chosen from among alcohol dehydrogenases (E.C. 1.1.1.1).

Without being limited, preferably those enzymes of microorganisms of the genus Aromatoleum (sometimes also referred to as Azoarcus) are available, such as, for example, Aromatoleum aromaticum, in particular strain EbN1.

Preferred enzymes with ADH activity comprise an amino acid sequence according to SEQ ID NO: 2.

According to the invention, "functional equivalents" of the ADHs specifically disclosed and their use in the process according to the invention are also included.

Within the framework of the present invention "functional equivalents" or analogs of enzymes specifically disclosed are polypeptides different from them, which also possess the desired biological activity, such as substrate specificity. Thus, for example, under "functional equivalents" are understood to be enzymes that reduce 3'-hydroxy-2-chloroacetophenone 2 to the corresponding alcohol R, (R) -3- (2-chloro-1-hydroxyethyl) phenol 3 and that they exhibit at least 50%, particularly preferably 75%, very particularly preferably 90% of the activity of an enzyme comprising one of the amino acid sequences cited under SEQ ID NO: 2.

According to the invention, "functional equivalents" in particular are also understood as mutants that in at least one sequence position of the amino acid sequences mentioned above, exhibit another amino acid different from that specifically mentioned, but nevertheless possess one of the Biological activities mentioned above. The "functional equivalents" thereby comprise the mutants obtainable by one or more additions, substitutions, deletions and / or reversals of amino acids, in which the aforementioned modifications can occur in any sequence position, as long as they lead to a mutant with the profile of properties according to the invention. In particular, functional equivalence is also given, when the pattern of reactivity between mutant and unmodified polypeptide qualitatively matches, that is, for example, the same substrates are transformed with different rates.

In the above sense the "functional equivalents" are also "precursors" of the polypeptides described as well as "functional derivatives" and "salts" of the polypeptides.

"Precursors" are in this respect natural or synthetic precursors of the polypeptides with the desired biological activity. Likewise, synthetic or natural precursors of the polypeptides without the desired biological activity are described.

The term "salts" means both salts of carboxyl groups and also acid addition salts of amino groups of the protein molecules according to the invention. The salts of carboxyl groups can be prepared in a manner known per se and comprise inorganic salts, such as sodium, calcium, ammonium, iron and zinc salts, as well as salts with organic bases, such as amines, such as triethanolamine, arginine. , lysine, piperidine and the like. Also included in the invention are acid addition salts, such as salts with mineral acids, such as hydrochloric acid or sulfuric acid and salts with organic acids, such as acetic acid and oxalic acid.

Likewise, "functional derivatives" of polypeptides according to the invention can be prepared in side functional groups of amino acids or at their N or C terminal ends with the aid of known techniques. Such derivatives comprise for example aliphatic esters of carboxylic acid groups, amides of carboxylic acid groups, obtainable by reaction with ammonia or a primary or secondary amine; N-acyl derivatives of free amino groups, prepared by reaction with acyl groups; or O-acyl derivatives of free hydroxyl groups, prepared by reaction with groups acyl.

"Functional equivalents" naturally also comprise polypeptides that are accessible from other organisms, as well as from naturally occurring variants. For example, by comparison of sequences, intervals of regions of homologous sequences are established and equivalent enzymes are determined following the specific procedures of the invention.

The "functional equivalents" also comprise fragments, preferably individual domains or sequence motifs of the polypeptides according to the invention, which exhibit, for example, the desired biological function.

"Functional equivalents" are also fusion proteins that exhibit one of the aforementioned polypeptide sequences or functional equivalents derived therefrom and at least one other functionally different heterologous sequence thereof, in terminal functional binding at N or C (ie, without essential mutual functional damage of the fusion profile area). Non-limiting examples of such sequences are for example peptides of

signal or enzymes.

In accordance with the invention, the "functional equivalents" included are homologs to the specifically disclosed proteins. These have at least 75% in particular at least 85%, such as 90, 91, 92, 93, 94, 95, 96, 97, 98 or 99%, of homology against one of the specifically disclosed amino acid sequences. A percentage homology of a homologous polypeptide according to the invention means in particular percent identity of the amino acid radicals, referred to the total length of one of the amino acid sequences specifically described herein.

"Identity" between two sequences in particular is understood as the identity of the radicals over the total sequence length in each case, in particular the identity that is calculated by comparison with the aid of Vector 10 NTI Suite 7.1 (Vector NTI Advance 10.3.0 , Invitrogen Corp.) (or software from the Informax (USA) company using the Clustal method (Higgins DG, Sharp PM. Fast and sensitive multiple sequence alignments on a microcomputer. Comput Appl. Biosci. April 1989; 5 (2): 151-1), by adjusting the following parameters:

Multiple alignment parameters:

Penalty for breach 10

15 Penalty for extension of gap 0.05

Penalty gap for gap separation 8 Penalty for gap separation off

Identity% by alignment delay 40

Specific residue gaps off

20 Hydrophilic residue gap off

Transition Weight 0

Paired alignment parameters:

FAST algorithm off

K-tuple size 1

25 Gap penalty 3

Window size 5

Number of best diagonals 5

In the case of a possible protein glycosylation, the "functional equivalents" according to the invention comprise proteins of the type referred to above in deglycosylated or glycosylated form, as well as modified forms obtainable by changes in the glycosylation pattern.

Homologs of the proteins or polypeptides according to the invention can be generated, by mutagenesis, for example by point mutation or shortening of the protein.

The homologs of the proteins according to the invention can be identified by discriminating combinatorial banks of mutants, such as shortening mutants. For example, a multicolored bank of protein variants can be generated by combinatorial mutagenesis on the nucleic acid plane, such as by enzymatic binding of a mixture of synthetic oligonucleotides. There is a multiplicity of procedures, which can be used for the preparation of banks of potential homologues from a degenerate oligonucleotide sequence. The chemical synthesis of a degenerated gene sequence can be executed in a DNA synthesis kit, and the synthetic gene can then be linked into a suitable vector of expression. The use of a degenerate gene phrase makes it possible to supply complete sequences in a mixture, which encodes the desired phrase into potential protein sequences. Methods for the synthesis of degenerate oligonucleotides are known to experts (for example Narang, SA (1983) Tetrahedron 39: 3; Itakura et al. (1984) Annu. Rev. Biochem. 53: 323; Itakura et al., ( 1984) Science 198: 1056; Ike et al. (1983) Nucleic Acids Res. 11: 477).

In the state of the art several techniques are known to discriminate gene products from combinatorial banks, which were prepared by point mutations or shortening, and to discriminate cDNA banks in

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gene products with a chosen property. These techniques conform to the rapid discrimination of gene banks that have been generated by combinatorial homologue mutagenesis according to the invention. The techniques most frequently used to discriminate large gene banks, which underlie a high performance analysis, include the cloning of gene banks into replicable expression vectors, transformation of suitable cells with the resulting vector bank and expression of combinatorial genes. Under conditions under which the detection of the desired activity facilitates the isolation of the vector encoding the gene, whose product was detected. Recursive-Ensemble-Mutagenese (REM), a technique that increases the frequency of functional mutants in banks, can be used in combination with discrimination tests, to identify counterparts (Arkin and Yourvan (1992) PNAS 89: 7811-7815 ; Delgrave et al. (1993) Protein Engineering 6 (3): 327-331).

4.2 Nucleic acid sequences encoding

In the context of the invention, the concepts "express" or "overexpression" describe the production or increase of the intracellular activity of one or more enzymes in a microorganism, which are encoded by the corresponding DNA. For this purpose, for example, a gene can be incorporated into an organism, replace a gene present with another gene, which increases the number of copies of the gene or genes, use a strong promoter or use a gene that codes for a corresponding enzyme with a high activity and these measures can be combined if necessary.

Particularly described herein are nucleic acid sequences that code for an enzyme with ADH activity. Nucleic acid sequences comprising a sequence according to SEQ ID NO: 1 are preferred; or nucleic acid sequences derived from amino acid sequences according to SEQ ID NO .: 2.

All nucleic acid sequences mentioned herein (single or double stranded DNA and RNA sequences, such as cDNA and mRNA) can be prepared in a manner known per se by chemical synthesis from nucleotide elements, such as by condensation of fragments of elements of the double helix of individual nucleic acid that overlap, complementary. The chemical synthesis of oligonucleotides can occur for example in a known manner, according to the phosphoamidite method (Voet, Voet, 2nd edition, Wiley Press New York, pages 896-897). In Sambrook et al. (1989), molecular Cloning: A laboratory manual, Cold Spring Harbor Laboratory Press, describes the addition of synthetic oligonucleotides and the filling of voids using the Klenow fragment of the DNA-polymerase binding reactions as well as general cloning procedures .

Also described herein are nucleic acid sequences (single and double stranded sequences of DNA and RNA, such as cDNA and mRNA), which code for one of the above polypeptides and their functional equivalents, which for example are accessible for example by use of artificial nucleotide analogs.

Both isolated nucleic acid molecules are described, which code for biologically active polypeptides or proteins or cuts thereof, as well as nucleic acid fragments, which can be used for example for use as hybridization probes or primers for the identification or amplification of nucleic acids that encode.

Nucleic acid molecules may also contain untranslated sequences from the 3 'and / or 5' end of the gene range that encodes.

Nucleic acid molecules complementary to the nucleotide sequences specifically described, or a cut of them, are also described.

The nucleotide sequences described herein make it possible to generate probes and primers, which can be used for the identification and / or cloning of homologous sequences in other types of cells and organisms. Such probes or primers usually comprise a nucleotide sequence range, which under "stringent" conditions (see below) hybridize at least about 12, preferably at least about 25, such as about 40, 50 or 75 consecutive nucleotides of a string in the sense of a nucleic acid sequence described herein, or a corresponding antisense string.

An "isolated" nucleic acid molecule is separated from other nucleic acid molecules, which are present in the natural source of the nucleic acid and may also be essentially free of other cell material or culture medium, when prepared by recombinant techniques, or be free of chemical precursors or other chemical substances, when it is synthesized by chemical means.

A nucleic acid molecule described herein can be isolated by means of standard molecular biology techniques and the sequence information provided in accordance with the invention. For example, cDNA can be isolated from a suitable cDNA bank, in which one of the complete sequences specifically disclosed or a section thereof is used as a hybridization probe, and standard hybridization techniques (as is

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described for example in Sambrook, J., Fritsch, E.F. and Maniatis, T. molecular Cloning: A Laboratory Manual. 2nd ed., Cold Spring Harbor Laboratory, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 1989). In addition, a nucleic acid molecule comprising one of the disclosed sequences or a section thereof is isolated, by polymerase chain reaction, in which the oligonucleotide primer that was prepared based on this sequence is used. The nucleic acid thus amplified can be cloned into a suitable vector and be characterized by DNA sequence analysis. The oligonucleotides according to the invention can also be prepared by standard synthesis procedures, for example with an automatic DNA synthesis apparatus.

The nucleic acid sequences described herein, such as SEQ ID No: 1 or derivatives thereof, homologues or parts of these sequences, are isolated, for example, with standard hybridization procedures or the PCR technique from suitable microorganisms, for example by genomic banks. or cDNA. These DNA sequences give hybridization under standard conditions with the sequences described herein. Short oligonucleotides are advantageously used for hybridization. But for hybridization, longer fragments of the nucleic acids described herein or the complete sequences can also be used. Depending on the nucleic acid used (oligonucleotide, longer fragment or complete sequence) or depending on which type of DNA or RNA nucleic acid is used for hybridization, these standard conditions vary. Thus, for example, the melting temperatures for DNA: DNA hybrids are 10 ° C lower than those of DNA: RNA hybrids of the same length.

Standard conditions are understood, for example depending on the nucleic acid, temperatures between 42 and 58 ° C in an aqueous buffer solution with a concentration between 0.1 to 5 x SSC (1 X SSC = 0.15 M NaCl, sodium citrate 15 mM, pH 7.2) or additionally in the presence of 50% formamide such as 42 ° C in 5 x SSC, 50% formamide. Advantageously, the hybridization conditions for DNA: DNA hybrids are at 0.1 x SSC and temperatures between about 20 ° C to 45 ° C, preferably between about 30 ° C to 45 ° C. For DNA: RNA hybrids the hybridization conditions are advantageously at 0.1 x SSC and temperatures between about 30 ° C to 55 ° C, preferably between about 45 ° C to 55 ° C. These temperatures indicated for hybridization are melting temperature values advantageously calculated for a nucleic acid with a length of approximately 100 nucleotides and a G + C content of 50% in the absence of formamide. Experimental conditions for DNA hybridization are described in relevant textbooks on genetics, such as Sambrook et al., "Molecular Cloning", Cold Spring Harbor Laboratory, 1989, and are calculated according to formulas known to experts, for example, depending on the length of the nucleic acid, the type of hybrid or the content of G + C. the expert can take other information for the hybridization of the following textbooks: Ausubel et al. (eds), 1985, Current Protocols in molecular Biology, John Wiley & Sons, New York; Hames and Higgins (eds), 1985, Nucleic Acids Hybridization: A Practical Approach, IRL Press at Oxford University Press, Oxford; Brown (ed), 1991, Essential molecular Biology: A Practical Approach, IRL Press at Oxford University Press, Oxford.

Derivatives of specifically disclosed or derivable nucleic acid sequences are also described herein.

Thus, other nucleic acid sequences described herein can be derived, for example, from SEQ ID NO: 1, and are differentiated therefrom by addition, substitution, insertion or elimination of one or more nucleotides, but also encode for polypeptides with the desired profile of properties.

Also described herein are those nucleic acid sequences that comprise so-called mute mutations or correspondingly to the codon use of an original organism or special host, are modified compared to a specifically mentioned sequence, as well as variants of natural occurrence thereof, such as splice variants or allele variants.

Also described are sequences obtainable by nucleotide substitutions that they retain (ie replace the amino acid in question with an amino acid of the same charge, size, polarity and / or solubility).

Also described herein are molecules derived by sequence polymorphisms of the specifically disclosed nucleic acids. These genetic polymorphisms may exist among individuals within a population, due to natural variation. These natural variations commonly cause a 1 to 5% variance in the nucleotide sequence of a gene.

Derivatives of the nucleic acid sequence described herein with the sequence SEQ ID NO: 1 are understood as, for example, allele variants that exhibit at least 40% homology on the amino acid derived plane, preferably at least 60% of Homology, very particularly preferably at least 80% homology over the entire sequence scope (with respect to homology at the amino acid level, refers to the previous embodiments of the polypeptides). On partial areas of the sequences, the homologies may be advantageously higher.

In addition, homologues of nucleic acid sequences are also understood as being derived, in particular from SEQ ID NO: 1, for example fungal or bacterial homologues, shortened sequences, DNA or string RNA

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individual of the DNA sequence that encodes and does not encode. Thus, for example, homologues of SEQ ID NO: 1 in the DNA plane have a homology of at least 40%, preferably at least 60%, particularly preferably at least 70%, very Particularly preferably at least 80% over the entire range of DNA indicated in SEQ ID NO: 1.

In addition, derivatives are understood as mergers with promoters. The promoters that precede the indicated nucleotide sequences may be modified by one or more nucleotide replacements, insertions, inversions and / or deletions, without impairing the functionality or effectiveness of the promoter. In addition, promoters can increase their effectiveness by modifying their sequence, or be completely exchanged for more effective promoters also from dissimilar organisms.

Variants are also understood as those whose nucleotide sequence was modified in the range of -1 to - 1000 bases upstream before the start codon or 0 to 1000 bases downstream after the stop codon, so that it changes, preferably increases, gene expression and / or protein expression.

In addition, nucleic acid sequences are also described, which hybridize with coding sequences mentioned above, under "severe conditions". These polynucleotides are detected in the screening of genomic banks or cDNA banks and if necessary they are propagated from them with suitable primers by means of PCR and then they are isolated, for example, with suitable probes. In addition, the polynucleotides described herein can also be synthesized chemically. This property is understood as the ability of a poly or oligonucleotide to bind under severe conditions to an almost complementary sequence, while under these conditions nonspecific bonds between non-complementary partners cease. For this, the sequences should be complementary in 70-100%, preferably in 90-100%. The property of the complementary sequences of being able to bind each other specifically, benefits for example in the technique of the northern spot or the southern spot or in the primer binding in PCR or RT-PCR. Oligonucleotides from a length of 30 base pairs are commonly used for this. Severe conditions are understood, for example, in the northern spot technique, to the use of a hot wash solution at 50-70 ° C, preferably 60-65 ° C, for example 0.1x SSC buffer with 0.1% SDS (20x SSC: 3M NaCl, 0.3M sodium citrate, pH 7.0) for non-specific elution of hybrid cDNA or oligonucleotide probes. In this regard they remain mutually linked, as mentioned above, only complementary nucleic acids to a large extent. The adjustment of severe conditions is known to the experts and is described for example in Ausubel et al., Current Protocols in molecular Biology, John Wiley & Sons, N.Y. (1989), 6.3.1-6.3.6 ..

4.3 Fragments used

Expression fragments are also used, which contain among the genetic controls of nucleic acid regulatory sequences, a nucleic acid sequence encoding an enzyme according to the invention; as well as vectors comprising at least one of these expression fragments.

Preferably such fragments comprise a promoter in 5 'upstream of the respective sequence encoding and in 3' downstream a termination sequence, as well as other current regulatory elements, and specifically in each case operatively linked with the sequence. that encodes

An "operative union" is understood as the sequential array of promoter, coding sequence, terminator and, where appropriate, other regulatory elements, so that each of the regulatory elements can satisfy how its function is intended in the expression of the sequence that encodes Examples of sequences that can be operatively linked, the target sequences as well as enhancers, polyadenylation signals and the like. Other regulatory elements include markers that can be selected, amplification signals, sources of replication and the like. For example, in Goeddel, Gene Expression Technology: Methods in Enzymology 185, Academic Press, San Diego, CA (1990) suitable regulatory sequences are described.

A nucleic acid fragment used in particular is the ADH with sequence SEQ ID NO: 1 and the derivatives and homologs thereof, as well as the nucleic acid sequences that can be derived from SEQ ID NO: 1, which were advantageously linked to operational or functional mode with one or several regulatory signals for modulation, for example elevation, of gene expression.

In addition to these regulatory sequences, the natural regulation of these sequences may still be present before the true structural genes and if necessary had been genetically modified, so that natural regulation was disabled and gene expression was increased. The nucleic acid fragment may also be constituted in a simple manner, that is, no additional regulatory signal was inserted before the coding sequence (such as SEQ ID NO: 1 or its counterparts) and the natural promoter was not removed with its regulation. Instead, the natural regulatory sequence mutates so that no more regulation occurs and gene expression rises.

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A preferred nucleic acid fragment also advantageously contains one or more of the aforementioned "enhancer" sequences, functionally linked with the promoter, which makes possible a high expression of the nucleic acid sequence. Also advantageous additional sequences, such as other regulatory elements or terminators, can be inserted at the 3 'end of the DNA sequences. The nucleic acids described herein may be present in one or several copies in the fragment. Other markers, such as antibiotic resistance or genes that complement auxotrophy, may be present in the fragment, if necessary for selection in the fragment.

Advantageous regulatory sequences for the process are present, for example, in promoters such as cos, tac, trp, tet, trp-tet, lpp, lac, lpp-lac, laclq T7, T5, T3, gal, trc, ara, rhaP ( rhaPBAD) SP6, lambda-PR or the lambda-PL promoter, which find advantageous application in gram-negative bacteria. Other advantageous regulatory sequences are present, for example, in the amy and SPO2 gram-positive promoters, in the yeast or fungal promoters ADC1, MFalpha, AC, P-60, CYC1, GAPDH, TEF, rp28, ADH. In this relationship, the promoters of pyruvate decarboxylase and methanoloxidase, for example Hansenula, are also advantageous. Artificial promoters can also be used for regulation.

The nucleic acid fragment is inserted for expression in a host organism, advantageously in a vector such as a plasmid or a phage, which makes possible optimal expression of the genes in the host. Vectors, apart from plasmids and phages, are also understood to be all other vectors known to the experts, therefore for example viruses, such as SV40, CMV, baculovirus and adenovirus, transposons, IS elements, plasmids, cosmids, and linear or circular DNA . These vectors can replicate autonomously in the host organism or replicate in a chromosomal manner. They are for example suitable plasmids in E. coli pLG338, pACYC184, pBR322, pUC18, pUC19, pKC30, pRep4, pHS1, pKK223-3, pDHE19.2, pHS2, pPLc236, pMBL24, pLG200, pUR290, pIN-III113 or pBdCl, in Streptomyces pIJ101, pIJ364, pIJ702 or pIJ361, in Bacillus pUB110, pC194 or pBD214, in Corynebacterium pSA77 or pAJ667, in fungi pALS1, pIL2 or pBB116, in yeast 2 and 6 in PEGL1 and PEGB1M plants pLGV23, pGHlac +, pBIN19, pAK2004 or pDH51. The plasmids mentioned represent a small selection of the possible plasmids. Other plasmids are well known to experts and can be taken, for example, from Cloning Vectors (Eds. Pouwels P. H. et al. Elsevier, Amsterdam-New York-Oxford, 1985, ISBN 0 444 904018).

Advantageously, the nucleic acid fragment contains for the expression of the other genes present, additionally still 3 'and / or 5' terminal regulatory sequences to raise the expression, which are chosen depending on the host organism selected and gene or genes. , for optimal expression.

These regulatory sequences should make possible focused gene expression and protein expression. This may mean, for example, depending on the host organism, that the gene is expressed or overexpressed just after induction, or that it is expressed and / or immediately overexpressed.

In this regard, the sequences or regulatory factors can have a positive influence and therefore preferably increase the gene expression of the introduced genes. In this way, a reinforcement of the regulatory elements can advantageously occur in the transcription plane, in which strong transcription signals are used as promoters and / or "enhancers". Apart from this, a translation reinforcement is also possible, in which the stability of the mRNA is improved, for example.

In another form of conditioning the vector, the vector containing the nucleic acid fragment or nucleic acid can be introduced into the microorganism, also advantageously in the form of a linear DNA and by heterologous or homologous recombination integrating it into the genome of the host organism . This linear DNA may consist of a linear transformed vector such as a plasmid or only the nucleic acid fragment or nucleic acid described herein.

For optimal expression of heterologous genes in organisms, it is advantageous to modify the nucleic acid sequences correspondingly to the specific "codon use" used in the organism. "Codon use" is easily determined by computer evaluations of other known genes of the organism in question.

The preparation of an expression cassette described herein occurs by fusion of a suitable promoter with a suitable nucleotide sequence that encodes, as well as a termination or polyadenylation signal. For this, established recombination and cloning techniques are used, as described for example in T. Maniatis, E.F. Fritsch and J. Sambrook, molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, Cold Spring Harbor, NY (1989) as well as in T.J. Silhavy, M.L. Berman and L.W. Enquist, Experiments with Gene Fusions, Cold Spring Harbor Laboratory, Cold Spring Harbor, NY (1984) and in Ausubel, F.M. et al., Current Protocols in molecular Biology, Greene Publishing Assoc. and Wiley Interscience (1987).

The recombinant nucleic acid fragment or gene fragment is inserted for expression in an organism

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suitable host, advantageously in a specific sector of the host, which makes possible optimal expression of the gene in the host. Vectors are well known to experts and can be taken, for example, from "Cloning Vectors" (Pouwels P. H. et al., Editor, Elsevier, Amsterdam-New York-Oxford, 1985).

4.4 Convenient guests

With the help of the vectors described here, recombinant microorganisms can be produced, which are transformed for example with at least one vector described herein and can be used for the production of the polypeptides used or for the execution of the enzymatic reaction according to the invention.

Advantageously, the recombinant fragments described above are incorporated and expressed in a suitable host system. In this regard, familiar cloning and transfection methods known by the expert, such as coprecipitation, fusion of protoplasts, electroevaporation, retroviral transfection and the like, are preferably used to express the nucleic acids mentioned in the respective expression system. For example in Current Protocols in molecular Biology, F. Ausubel et al., Editor, Wiley Interscience, New York 1997, or Sambrook et al. Molecular Cloning: A Laboratory Manual. 2nd ed., Cold Spring Harbor Laboratory, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 1989 describes suitable systems.

Homologous recombinant microorganisms can also be prepared. For this purpose, a vector is prepared, which contains at least a section of a gene described herein or a coding sequence, in which case at least one amino acid elimination, addition or substitution was incorporated, to modify the sequence, for example, interrupt it in a functional way ("genetic blocking" vector). The incorporated sequence may for example also be a homologue of a relative microorganism or be derived from a mammalian, yeast or insect source. The vector used for homologous recombination can alternatively be conditioned so that the endogenous gene mutates by homologous recombination or is modified in another way, although it still encodes the functional protein (for example the upstream regulatory zone can be modified, so that which is modified by the expression of the endogenous protein). The modified gene cut is in the homologous recombination vector. The construction of suitable vectors to homologous recombination is described for example in Thomas, K.R. and Capecchi, M.R. (1987) Cell 51: 503.

As recombinant host organisms for the nucleic acids described herein or the nucleic acid fragment, all prokaryotic or eukaryotic organisms come into consideration in principle. Advantageously, microorganisms such as bacteria, fungi or yeasts are used as host organisms. Advantageously, gram-positive or gram-negative bacteria are used, preferably bacteria of the Enterobacteriaceae, Pseudomonadaceae, Rhizobiaceae, Streptomycetaceae, Bacillaceae or Nocardiaceae families, particularly preferably Escherichia, Pseudomonas, Streptomyces, Nocardia, Burkholderia, Salmonella genera , Agrobacterium, Bacillus or Rhodococcus. The genus and type Escherichia coli is very particularly preferred. In addition other advantageous bacteria are in the group of alpha-proteobacteria, beta-proteobacteria or gamma-proteobacteria.

In this regard, the host organism or host organisms preferably contain at least one of the described nucleic acid fragments, nucleic acid sequences or vectors, which code for an ADH enzyme.

The organisms used in the process according to the invention are cultured or reared, depending on the host organism in a manner known to the expert. Microorganisms are generally grown in a liquid medium that contains a carbon source usually in the form of sugars, a nitrogen source usually in the form of organic nitrogen sources such as yeast extract or salts such as ammonium sulfate, trace elements such as salts of iron, manganese, magnesium, at temperatures between 0 ° C and 100 ° C, preferably between 10 ° C to 60 ° C under exposure to gaseous oxygen. In this regard, the pH of the nutrient liquid can be maintained at a fixed value, that is, to be regulated or not during propagation. Propagation can occur in "batch" mode, in "semi-batch" mode or continuously. Nutrients can be placed prior to the start of fermentation or supplied semi-continuously or continuously.

The ketone to be reacted can be added directly to the propagation or advantageously after the propagation.

Enzymes can be isolated from organisms or used as a crude extract for the reaction.

The host organisms contain an enzymatic activity for example of 1 U / I, such as ADH activity, preferably 100 U / I, particularly preferably greater than 1000 U / I.

4.5 Recombinant enzyme preparation:

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The enzymes used are also accessible by recombinant preparation, in which a microorganism that produces these enzymes is cultured, if necessary the expression of the polypeptide is induced and this is isolated from the culture. The polypeptides can also be produced on an industrial scale, if desired.

The recombinant microorganism can be cultured and fermented according to known procedures. Bacteria can be multiplied for example in TB or LB medium and at a temperature of 20 to 40 ° C and a pH value of 6 to 9. For example in T. Maniatis, E.F. Fritsch and J. Sambrook, molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, Cold Spring Harbor, NY (1989) describe in detail the suitable culture conditions.

The cells are then macerated, in case the polypeptide is not secreted in the culture medium, and the product is obtained according to known protein isolation procedures, from the lysis product. At choice, the cells can be subjected to high frequency ultrasound digestion, by high pressure, such as in a French pressure cell, by osmolysis, by the action of detergents, lithic enzymes or organic solvents, by homogenizers or by combination of several of the procedures cited.

Purification of the polypeptide can be achieved with known chromatographic procedures, such as molecular sieve chromatography (gel filtration), such as Q-Sepharose chromatography, ion exchange chromatography and hydrophobic chromatography, as well as with other common procedures such as ultrafiltration, crystallization, precipitation by salts, dialysis and native gel electrophoresis. For example, Cooper, F. G., Biochemische Arbeitsmethoden, Verlag Walter de Gruyter, Berlin, New York or Scopes, R., Protein Purification, Springer Verlag, New York, Heidelberg, Berlin describe suitable procedures.

For the isolation of the recombinant protein, it may be advantageous to use vector or oligonucleotide systems, which lengthen the cDNA in certain nucleotide sequences and thereby encode modified polypeptides or fusion proteins, which serve, for example, for simpler purification. Such suitable modifications are for example called "beacons" that act as an anchor, such as the modification or epitope known as a hexa-histidine anchor, which can be recognized as antibody antigens (described for example in Harlow, E. and Lane, D., 1988, Antibodies: A Laboratory Manual, Cold Spring Harbor (NY) Press). These anchors can be used to bind the protein to a solid support, such as a polymer matrix, which can be packed, for example, in a chromatographic column, or it can be used in a microtiter plate or other support.

Simultaneously these anchors can also be used for protein recognition. For the recognition of proteins, current markers, such as fluorescence dyes, enzyme markers, which after reaction with a substrate form a detectable reaction product, or radioactive markers, alone or in combination with the anchors, can also be used to form the protein derivative.

4.6 Carrying out step b) of the process according to the invention for the preparation of optically active alcohols

The enzymes used can be used in the process step according to the invention, as a free or immobilized enzyme.

The process step according to the invention is advantageously performed at a temperature between 0 ° C and 60 ° C, preferably between 10 ° C and 40 ° C, particularly preferably between 15 ° C and 35 ° C.

The pH value during the process step according to the invention is advantageously maintained between pH 4 and 12, preferably between pH 4.5 and 9, particularly preferably between pH 5 and 8.

For the process according to the invention, growing cells containing nucleic acids, nucleic acid fragments or vectors according to the invention can be used. Resting or macerated cells can also be used. For example, macerated cells are understood to mean cells that by means of a treatment with, for example, solvents have been converted to porous, or cells that were broken by an enzymatic treatment, by a mechanical treatment (for example French press or ultrasound) or by other method. The crude extracts thus obtained are suitable for the process according to the invention. Purified or unpurified enzymes can also be used for the process. Likewise, immobilized microorganisms or enzymes are suitable.

If free organisms or enzymes are used for the process according to the invention, then these are conveniently separated before extraction, for example by filtration or centrifugation.

If a biphasic reaction medium (aqueous / organic) is used, then the isolation of the product is facilitated, since the valuable product can be dissolved preferably in the organic phase. For example, an essentially water-miscible solvent, such as an ether, is used in particular to form the system

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biphasic

If, on the contrary, in the enzymatic stage of the process a single-phase reaction medium is used, then the product prepared from the aqueous reaction solution is obtained, by extraction or distillation or advantageously by extraction and distillation. The extraction can be repeated several times to increase the yield. Examples of suitable extraction agents are solvents, such as toluene, methylene chloride, butyl acetate, diisopropyl ether, benzene, MTBE or acetic ester, without being limited thereto.

After concentrating the organic phase, the products can be obtained as a rule with good chemical purities, that is to say with a chemical purity greater than 80%, 90%, 95% or 99%. After extraction, the organic phase can be concentrated with the product, but also only partially, and the product crystallized out. For this, the solution is advantageously cooled to a temperature of 0 ° C to 10 ° C. Crystallization can also occur directly from the organic solution or from an aqueous solution. The product separated by crystallization can be collected once more in the same or another solvent, for a new crystallization, and be crystallized once more. The subsequent enantiomer purity of the product can be increased further by the subsequent crystallization carried out at least once, if necessary.

In the aforementioned types of processing, the product of the process according to the invention is isolated with yields of 60 to 100%, preferably 80 to 100%, particularly preferably 90 to 100%, based on the substrate used for the reaction. The isolated product is distinguished by a high chemical purity of> 90%, preferably> 95%, particularly preferably> 98%. In addition, the products have a high enantiomer purity which, if necessary, can be further advantageously enhanced by crystallization.

The process according to the invention can be operated in batch mode, semi-batch mode or continuously.

Execution of the procedure can take place advantageously in bioreactors, as described for example in Biotechnology, volume 3, 2nd edition, Rehm et al., (1993) in particular chapter II.

The above description and the following examples serve only to clarify the invention. Likewise, the numerous possible modifications evident to the expert are possible.

Experimental part:

Example 1: synthesis of HCAP, 2 (in ethyl acetate):

In a 6000 ml mini-plant reactor with agitator impeller, flow deflector, thermometer and drip funnel, 435.68g (3.20 mol) of 3-hydroxyacetophenone in 410.111g (12.80 mol) of methanol and 1200 ml of ethyl acetate. To this solution are added dropwise at 20-30 ° C under cooling 691.05g (5.12 mol) of sulfuryl chloride over a period of 2h. After the drip addition the charge is stirred for an additional hour at room temperature. After that the charge is hydrolyzed at room temperature with 1600 ml of H2O and the resulting biphasic mixture is separated. Extraction to the aqueous phase is performed once with 800 ml of ethyl acetate. Of the combined organic phases, methanol and ethyl acetate are distilled off by a distillation bridge. Simultaneously, 1880 ml of isopropanol are added to the bottom of the distillation. 2462.5g of a 17.3% solution in isopropanol are obtained, of the valuable product which corresponds to a content of 426 g (2.51 mol). With this, the yield is 78%.

Example 2: synthesis of HCAP, 2 (in dichloromethane):

In a 2000 ml mini-plant reactor with stirrer impeller, flow deflector, thermometer and drip funnel, 204.23g (1.50 mol) of 3-hydroxyacetophenone are placed in 192.24g (6.00 mol) of methanol and 1050ml of CH2O2. To this solution are added dropwise at 20-30 ° C under cooling 283.44g (2.10 mol) of sulfuryl chloride over a period of 2 hours. After the drip addition, the charge is stirred for an additional hour at room temperature.

After that the charge is hydrolyzed at room temperature with 400 ml of H2O and the resulting biphasic mixture is separated. After phase separation, the organic phase, methanol and CH2Cl2 are distilled off under normal pressure by a distillation bridge. Simultaneously 880ml of isopropanol is added dropwise with the same speed. 837.78g of a 25.7% solution in isopropanol of the valuable product are obtained, which corresponds to a content of 215 g (1.26 mol). With this, the yield was 84%.

Example 3: synthesis of HCPE, 3:

A ketone 2 prepared as in Example 1 or 2 is reduced, by biocatalytic route to R-3. For this they dissolve

Claims (14)

5 10 fifteen twenty 25 30 35 1. Procedure for the preparation of optically active substituted alcohols of formula IV image 1 in which Cyc represents a mono or polynuclear ring, saturated or unsaturated, carbocyclic or heterocyclic, where appropriate substituted once or several times, which carries at least one free hydroxyl group, and R1 and R2 independently of each other represent H or a given alkyl radical the case replaced once or several times; or of salts of this compound; in each case in the form of pure stereoisomer or as a mixture of stereoisomers, in which a) a ketone of the formula I (I) aliphatic alcohol to give a halogenated compound (II) in which Cyc has the meanings indicated above and Hal represents a halogen atom; b) the compound of the formula II thus obtained is reduced enzymatically using an enzyme chosen from among alcohol dehydrogenases (ADH) (E.C. 1.1.1.1), to give the alcohol of the formula III OH image2 in which Cyc and Hal have the meanings indicated above; Y c) the alcohol of the formula III thus obtained reacts with an amine of the formula HNR1R2, in which R1 and R2 have the meanings indicated above; until giving the compound of the formula IV. 2. The method according to claim 1, wherein the reaction of step a) occurs in the presence of 1 to 10 molar equivalents of alcohol per mole of alkane of formula I. 3. Method according to one of the preceding claims, wherein the chemical reaction of step c) occurs in solution in an open or cyclic chain ether. 4. The method according to claim 1, wherein the alcohol dehydrogenase is chosen from among enzymes of microorganisms of the genus Aromatoleum. 5. Method according to one of the preceding claims, wherein the enzyme for step b) is chosen from among enzymes that possess a polypeptide sequence, which is chosen from (i) SEQ ID NO: 2 or (ii) sequences in which up to 25% of the amino acid radicals are modified with respect to SEQ ID NO: 2 by addition, elimination, insertion, substitution, inversion or a combination thereof, and / or that still exhibits at least 50% of the enzymatic activity of SEQ ID NO: 2. Cyc TO in which Cyc has the meanings indicated above, reacts with a halogenating agent in the presence of one of the formula II image3 5 10 fifteen twenty 25 Method according to one of the preceding claims, in which the reaction of step b) is carried out by adding reduction equivalents and, if necessary, the reduction equivalents consumed in the reaction are regenerated. 7. Method according to claim 6, wherein the regeneration is carried out by enzymatic, electrochemical or electroenzymatic route. 8. The method according to claim 7, wherein the regeneration occurs enzymatically and the enzyme it regenerates is chosen from ADH and dehydrogenases other than ADH. 9. Method according to one of the preceding claims, wherein the reaction of step b) occurs in the presence of a microorganism which expresses ADH naturally or recombinantly, or in the presence of a fraction derived therefrom, which contains ADH, or in the presence of an extract derived from it that contains ADH. 10. Method according to one of the preceding claims, wherein the reaction of step b) occurs in the presence of a microorganism that produces ADH, which is chosen from among bacteria of the Enterobacteriaceae, Pseudomonadaceae, Rhizobiaceae, Lactobacillaceae, Streptomycetaceae, Rhodococcaceae, Rhodociclaceae and Nocardiaceae, or in the presence of a fraction or an extract derived from them that has ADH. 11. A method according to claim 10, wherein the microorganism is a recombinant microorganism, which is transformed with a nucleic acid fragment, which encodes an alcohol dehydrogenase according to the definition of one of claims 4 to 6. 12. Method according to one of the preceding claims, wherein step b) is executed in a biphasic liquid reaction medium. 13. Method according to claim 12, wherein an aqueous-organic reaction medium is used, wherein both the reagent of the formula II and also the product of the formula III dissolve better in the organic phase than in the aqueous phase 14. Use of an alcohol dehydrogenase according to the definition of one of claims 1 to 6 or a microorganism that produces this enzyme, for the preparation of a compound of formula IV.